| Bacterial artificial chromosome (BAC) libraries with large genomic DNA inserts provide an invaluable tool in genomics studies including genome physical mapping, map-based cloning, genome sequencing and development of new molecualr markers. For the deep researches on cotton gemome, it is necessary for constructing more BAC libraries with large insert size and high coverage.Based on the method of Zhang HB, some modifications were made to meet the need of BAC library construction of cotton. A high-efficient technology system of BAC library constrcution was established, including prepartion of high-quality megabase-size DNA, determination of optimal partial digestion conditions, size selection, ligation and transformation.Utilizing improved methods, taking pIndigoBAC-5 as vector, two BAC libraries were constructed for G. arboreum L. (diploid species, genome type A2A2) and G.. hirsutum L. (tetraploid sepecies, genome type AADD). These new BAC libraries, combined with the developed cotton libraries, will provide sufficient clone resources for comprehensive genome research of the species.We have constructed a cotton BAC library using Gossypium arboreum L. cv. Jianglingzhongmian, an elite A-genome germplasm line. The BAC library contains 123,648 clones stored in 322 384-well plates cmprising 7.2 haploid genome equivalents based on a A genome size of 1690Mb. A random sampling of 103 BACs indicated an average insert length of 100.2 kb with a range of 30 to 190 kb, and less than 1% of the BACs do not contain inserts. Six clones were randomly selected from the library to determine the stability of BAC clones. There existed no different fingerprints of 0 and 100 generations of each clone digested with restriction enzyme NotⅠand HindⅢrespectively. This indicated that a single BAC clone could be sustained at least for 100 generations. For facilitating BAC library screening, the whole library was arranged in two levels of pools (row pools. RPs:Superpools. SPs) allowing screening with various PCR-based markers.The ordered array of the pooled BAC clones was screened and the BAC clones containing the GaMYB7 gene were identified. With the BAC-DNA serving as template for the first round of TAIL-PCR, we successfully obtained a sequence 1566 base pairs in length upstream of the ATG start codon of the GaMYB7 gene. Cis-acting element prediction results showed the basic sequence structure consisted of identified putative core promoter elements and other upstream promoter elements, including light response elements, auxin and gibberellin response elements. In transient assays with cotton ovules cultured in vitro, both this promoter sequence and a series of truncations could drive theβ-glucuronidase reporter gene (GUS) specifically in ovules and fibers. Therefore, the promoter is considered to be useful expression element for deep researches on molecular mechanisms of cotton fiber development, modification of fiber quality and expression of foreign genes in fiber.Another BAC library has been constructed for upland cotton using its genetic standard line TM-1 as material. The library consists of 170,880 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 8.6 haploid genome equivalents based on an AD genome size of 2,425 Mb. Clones were stored in 455 384-well plates and arrayed into multiplex pools for rapid and reliable library screening. Column-Pool library (CP1-CP19) consist of 19 384-well plate with 24 individual clones each well. Total 456 super-pools were stored in two 384-well plate with 384(24×16) clones each well.114 giant-pools were mixed from super-pools with 1536(24×16×4) clones each well. It needs 158 PCR reactions to hit a positive clone in the library.Chromosome 12 and 26 are two homoeologous chromosomes. By far,some important gene loci have been mapped on these two chromosomes. For isolating these important agricultural-related genes via map-based cloning, it is very important to construct BAC-based physical map of these two chromosomes. The BAC library of TM-1 was screened using the markers locolized on the chromosome 12 and 26 according to the genetic map. Correspondingly,607 positive clones were identified giving an average 4 positive signals per marker (range 1-16). Total 68 clones were shared by these two chromosomes.21 groups (about 49 markers) were shared the common clones. By conparsion, we found the markers sharing the common clones were located nearly on the genetic linkage map, which confirming the accuracy of the genetic map. Although some markers such as NAU3294 and NAU3293, BNL1673 and NAU1463, NAU4905 and NAU3905, NAU3441 and NAU3442 were mapped on the different chromosmes, they shared the common clones. It indicated the loci sites of the markers which sharing the same clones might be the homoeologous regions.BAC-DNA was prepared from total 380 positive clones (removing redundant clones from 607 clones) and digested by restriction enzyme HindⅢfor obtaining its fingerprint map. Through the analysis using Image 3.10b and FPCV8.5 software, the primary contigs were constructed. |
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